Medical diagnostic tools typically use classical biochemical techniques that involve bulky and expensive equipment such as spectrophotometry, gas chromatography (GC), mass spectrometry (MS), high-performance liquid chromatography (HPLC), paper and thin-layer chromatography (PC and TLC), and electrophoretic techniques coupled with fluorescence detection techniques. These standard analytical tools work effectively and efficiently. However, the tools are expensive and require costly consumables, sample throughput, and experienced and skilled operators. These drawbacks hinder rapid, inexpensive, and in-situ diagnosis of health-care. Such methods often require tedious and laborious processes. Therefore these tools are mostly used as confirmatory tools for the presumptive positive samples that are initially screened by some kinds of assay techniques.
Quantitative immunoassay techniques pose similar problems. The performance of quantitative immunoassay analysis is largely restricted to centralised laboratories because of the need for long assay times, and for relatively complex, bulky and expensive equipment, as well as highly-trained operators. Thus the analysis is performed far from the patients whose samples are measured. If a wider range of immunoassays could be run in a simpler fashion, more inexpensively and at the point of care or in home health care, the health of large numbers of patients could be improved annually.
Optical biosensors are one of the major types of biosensors to have been exploited for immunoassay applications due to the advantages they can offer, such as improved sensitivity, simplicity and immunity to electromagnetic wave interferences. Many types of optical techniques are commonly used for biosensing applications. Fluorescence-based sensors are perhaps the most highly developed due to their high sensitivity, versatility, accuracy and fairly good selectivity. Fluorescence methods are also very suitable for miniaturisation. The current focus in this area is to measure/detect fluorescently-labelled analytes inside a microfluidic channel by focusing an excitation light source onto a sample inside a microchannel and collecting the fluorescence emission of the sample using a set of complex lenses, mirrors, and optical filters. As a result, a fluorescence signal from the microfluidic substrate may enter the detection system giving rise to a strong but unwanted fluorescence noise. The fluorescence response from the analyte of interest is often rather weak due to the low analyte concentration. As a consequence, fluorescence noise due to the fluorescence of the substrate may suppress the wanted fluorescence signal from the analyte of interest. For early detection of diseases, biomarker concentrations are always low at the early stages of any diseases. Present point-of-care systems have limits in detecting low analyte concentration typical for early detection of diseases.
Two approaches commonly used to mitigate the effects of this are:    1) Incorporation of a confocal fluorescence microscope which can block the signals not from thin layer within which the sample resides. This technique works reasonably well, but it requires bulky, expensive and complicated optics.    2) A material with no or low fluorescence properties is selected as the substrate material. Optical grade glass and silica are commonly used for this purpose, since these materials have low autofluorescence when they are excited by light within visible wavelengths. However, these materials are relatively expensive and fabrication of microfluidic channels using these materials requires time-consuming photomask generation, photolithography and etching processes. As a consequence, the microfluidic chip made from optical grade glass or silica will be relatively expensive.
Candidate inexpensive materials considered as being suitable for use as substrate materials are polymer-based materials, such as polymethyl-methacrylate (PMMA), polycarbonate and Mylar. In addition, microfluidic channels using polymeric materials can be easily fabricated by moulding, embossing, casting or ablation processes. Complex models of microchannels in polymer sheets have been fabricated in less than an hour using a direct-write laser system. However, these materials exhibit relatively high autofluorescence signals which in turn hinder their use for low analyte concentration detection. The intensity of fluorescence background signal from the polymeric materials can be two orders of magnitude higher than fluorescence signal of sample within the microfluidic channel. Hence, utilisation of a polymeric microfluidic chip requires a technique that can resolve and eliminate the auto-fluorescence background noise of polymeric materials.
A background discussion of this technology reveals that when a fluorescent material is excited by a short pulse from an excitation light source, the material will fluoresce in such a way that its intensity decays exponentially with time. The time for the fluorescent intensity of the material to decay to 1/e of the initial intensity at t=0 is called the fluorescent lifetime of the material. In general, due to the chemical composition of the materials, different materials will fluoresce with different lifetimes. In the frequency domain, when the material is excited by a sinusoidally modulated light source, a sinusoidal fluorescence signal will be generated with a frequency that is identical to the frequency of the excitation light source but with its phase shifted with respect to the excitation light. Hence if two fluorescent materials are excited using a sinusoidal light source, two signals will be detected, the phases of which will vary depending on their respective fluorescent lifetimes.
When light, which is modulated to a frequency fmod is incident on the surface of the fluorescently labelled sample inside the microfluidic channel made of polymers, two sets of signals or signal components are produced, assuming that the excitation signal has already been filtered off. These signals/components are (1) the fluorescence signal component emitted by the labelled analyte of interest and (2) the fluorescence background noise signal/component emitted by the substrate. These two sets of signals will have the same modulating frequency as the incident light source, but in general, will be at different phase and amplitude with respect to the incident oscillating light source. This is illustrated in FIG. 1 illustrating the voltage-time characteristics 100 of the two signals, and is due to the difference in the different fluorescence lifetimes of the two polymers. As the analyte of interest immobilised within a microfluidic channel may be very low in concentration, it will form the weaker of the two signals. This weaker signal 102 is denoted as ys(t)=As sin(ωt+φs) where ω=2πfmod. The stronger signal 104 is the unwanted signal, as it represents the fluorescence background noise from the substrate. This is denoted as yn(t)=An sin(ωt+φn). Since, in general, the fluorescence signals from the analyte of interest and the substrate have different fluorescence lifetimes, there exists a phase difference between ys(t) and yn(t). The phase difference of the two signals with respect to the incident signal are denoted as φs and φn respectively. Given this, the detection of the weaker fluorescence signal of interest ys (t), is clearly problematic.
Commonly-assigned International Patent Publication No. WO2007/040459 discloses several techniques in this field. In at least one of these techniques, an output signal from the system which represents the wanted sample fluorescence signal is given by
            A      s        2    ⁢      sin    ⁡          (      Δϕ      )      where As is the amplitude of the signal and Δφ is the phase difference between the phase of the wanted sample fluorescence signal and the phase of the unwanted substrate fluorescence signal. An example of this technique is illustrated in circuit 20 of the appended FIG. 2, but a detailed description of the operation of circuit 20 is given in WO2007/040459.
The signal detected by the photodetector III of FIG. 2 is given asytotal=As sin(ωt+φs)+An sin(ωt+φn)To eliminate the noise signal, another signal q(t)=Ax cos(ωt+φn) is generated, which is orthogonal to the noise signal component An, using a phase delay circuit.
Multiplying these two signals, one obtains,
                                          x            q                    ⁡                      (            t            )                          =                                            y              total                        ⁡                          (              t              )                                ×                      q            ⁡                          (              t              )                                                              =                                                                              A                  s                                ⁢                                  A                  x                                            2                        ⁢                          sin              ⁡                              (                                                      ϕ                    s                                    -                                      ϕ                    n                                                  )                                              +                                                                      A                  s                                ⁢                                  A                  x                                            2                        ⁢                          sin              ⁡                              (                                                      2                    ⁢                    ω                    ⁢                                                                                  ⁢                    t                                    +                                      ϕ                    s                                    +                                      ϕ                    n                                                  )                                              +                                                                      A                  n                                ⁢                                  A                  x                                            2                        ⁢                          sin              ⁡                              (                                                      2                    ⁢                    ω                    ⁢                                                                                  ⁢                    t                                    +                                      2                    ⁢                                          ϕ                      n                                                                      )                                                        
                    A        s            ⁢              A        x              2    ⁢      sin    ⁡          (                        2          ⁢          ω          ⁢                                          ⁢          t                +                  ϕ          s                +                  ϕ          n                    )        ⁢          ⁢  and  ⁢          ⁢                    A        n            ⁢              A        x              2    ⁢      sin    ⁡          (                        2          ⁢          ω          ⁢                                          ⁢          t                +                  2          ⁢                      ϕ            n                              )      are eliminated by the low pass filter leaving
                              A          s                ⁢                  A          x                    2        ⁢          sin      ⁡              (                              ϕ            s                    +                      ϕ            n                          )              ,which is a DC signal that is dependent on the phase difference φs−φn.
                    A        s            ⁢              A        x              2    ⁢      sin    ⁡          (                        ϕ          s                +                  ϕ          n                    )      is maximum when φs−φn is 90°.
The phase difference is dependent on fluorescence lifetimes between the signal As and the noise An as well as the modulation frequency of the excitation light source I. As an example, for fluorescein and mylar, the fluorescent lifetime difference is 1 ns and to generate a 90° phase difference, the modulation frequency required for light source I is 250 MHz. Such high frequencies can be achieved using laser diodes but not LEDs. The dependence of the phase difference on the modulating frequency is not in direct proportion. In general, it is not possible to obtain a 90° phase shift. Depending on the samples, there will be a certain frequency at which the phase difference is a maximum. That will be the optimal operating frequency for that system. The shorter the difference in lifetimes, the higher is this frequency.
The DC output obtained by circuit 20 is
                    A        s            ⁢              A        x              2    .